Association of The Antitumoral Effect of
Bursera Fagaroides with The Inhibition of
Kidney Ornithine Decarboxylase in The L5178y Lymphoma Model
Ramón Reynoso-Orozco1, Anne Santerre1*, Jorge Iván Delgado-Saucedo2, Salvador Velázquez-Magaña1 and Ana María Puebla-Pérez2
1Department of Cellular and Molecular Biology, Laboratory of Molecular Biomarkers in Biomedicine and Ecology, University of Guadalajara, México
2Department of Pharmacobiology, University of Guadalajara, México
Submission: May 25, 2020; Published: August 07, 2020
*Corresponding author: Anne Santerre, Centro Universitario De Ciencias Biologicas Y Agropecuarias Camino Ramón Padilla Sánchez No. 2100, Las Agujas, Nextipac, Zapopan, Jalisco, México
How to cite this article: Ramón R-O, Anne S, Jorge I D-S, Salvador V-M, Ana M P-P. Association of The Antitumoral Effect of Bursera Fagaroides with The
Inhibition of Kidney Ornithine Decarboxylase in The L5178y Lymphoma Model. Nutri Food Sci Int J. 2020. 10(3): 555786. DOI: 10.19080/NFSIJ.2020.10.555786.
Ornithine decarboxylase and polyamines overexpression are key events for cancer growth, and potential markers of cancer outcome, as well as critical targets for cancer prevention or therapy. Bursera Fagaroides is a native plant to Mexico with antitumoral properties in the BALB/c mice L5178Y lymphoma model; however, its mechanism of action is not fully understood.
Thus, to better comprehend the antitumoral effect of B. fagaroides in this model, we evaluated ornithine decarboxylase activity, polyamines profile, total proteins, and tumor growth.
Lymphoma was induced by a single intraperitoneal injection of 2 x 104 L5178Y cells. The hydroalcoholic plant extract was given, daily, intraperitoneal or orally, at 100 mg/Kg body weight for 15 days. Kidney ornithine decarboxylase activity was determined by using a specific radiometric test, polyamines levels by liquid chromatography, and total proteins with the Bradford assay.
Experimental data showed that kidney ornithine decarboxylase activity increased in L5178Y mice compared to healthy mice. Remarkably, the intraperitoneal administration of B. fagaroides induced an 87% drop of kidney ornithine decarboxylase activity compared to untreated L5178Y mice. The oral administration of the plant extract did not show this positive effect. Biologically active polyamines: spermidine and spermine, levels in the kidney were unchanged between groups.
These data highlight that ornithine decarboxylase activity is a biomarker of L5178Y lymphoma growth in BALB/c mice, providing a model of great potential to search for new antitumoral compounds. In this model, the antitumoral property of B. fagaroides involves the inhibition of ornithine decarboxylase activity and the maintenance of polyamines homeostasis.
Polyamines (PA), mainly putrescine, spermidine, and spermine are small aliphatic basic molecules that play important roles in cell physiology . They are distributed ubiquitously in all cells and organs and are generally abundant in rapidly dividing cells. PA are necessary for the completion of the cell cycle of normal cells as well as hyperproliferative cancer cells [2-10]. PA homeostasis is the product of complex regulatory mechanisms and signal transduction events that involve de novo biosynthesis, active
uptake, and efflux, and complex feedback mechanisms [11-13]. Ornithine decarboxylase (ODC) is the first rate-limiting enzyme for the PA biosynthetic pathway. Although mandatory for PA synthesis, ODC is expressed in small amounts in almost all normal cells and its half-life is one of the shortest of mammalian enzymes (10-30 min), allowing an efficient regulation of its activity and PA biosynthesis . PA levels change rapidly upon modification in the rates of synthesis or degradation of ODC (Graphical abstract).
Because ODC overexpression participates in the transformation
of a normal cell to a malignant one, it is a critical target for the
prevention and therapy of cancer and other hyperproliferative
diseases [6,15]. For example, 2-difluoro-methylornithine (DFMO)
is a synthetic molecule that irreversibly binds to ODC active
sites, inhibiting enzymatic activity and PA biosynthesis [15,16].
Its effects have been documented in animal models and several
human clinical trials, especially for hyperproliferative and
infective diseases , as well as colon cancer chemoprevention
and neuroblastoma [17-19]; however, its clinical use may present
cytostatic and noncytotoxic secondary effects . Thus, on an
intention to treat hyperproliferative diseases, it is important to
pursuit new molecules of natural or synthetic origin that target
ODC activity and PA metabolism.
Bursera Fagaroides, a native plant from Mexico, traditionally
used in religious and medicinal contexts, presents antitumoral
effects in BALB/c mice with L5178Y lymphoma . In a previous
study, we showed the utility of urinary PA, especially the diamine
putrescine as a biomarker of L5178Y lymphoma growth. The
antitumoral activity of B. fagaroides was associated with a decrease
in putrescine levels in the urine of treated animals compared to
the urine of untreated ones . However, no decisive conclusion
could be drawn on the mechanisms of action of the extracts, and if
it targets ODC activity and PA metabolism.
The main goals of the present study were two-fold: to evaluate
ODC activity and PA levels in the BALB/c L5178Y lymphoma
model, and to determine the contribution of both parameters to
the antitumoral effect of B. fagaroides.
Bursera Fagaroides was originally collected during January
1995 in Michoacán State, Mexico (19°40′25″N 101°23′36″O).
Identification was performed both at the Instituto Mexicano
del Seguro Social´s Herbarium, by A. Aguilar (registry number
IMSSM-12051) and by Jaqueline Reynoso-Dueñas, Institute of
Botany, University of Guadalajara (registry number IBUG 140748)
as previously reported . The hydroalcoholic extract of B.
fagaroides was prepared according to Puebla-Pérez et al. , and
administered to BALB/c mice intraperitoneally (i.p.) or orally at
100 mg/Kg body weight, daily and for 15 days, starting 24 h after
tumor cells inoculation .
Thirty male adult BALB/c mice [haplotype] H2d of six to eight
weeks of age were used. Animals were maintained in a specific
pathogen-free animal care facility in a regulated environment
(22±1°C, 50–60% relative humidity, and 12-h light cycle). All mice
were fed a commercial diet Lab Chow (Purina Lab Chow–Mexico)
with autoclaved tap water ad libitum. All experimental procedures
were performed following the protocols approved by the Research
Center’s animal care committee and in compliance with national
guidelines on animal welfare (Official Mexican standard NOM-
062-ZOO-1999, Technical specifications for production, care and
use of laboratory animals) .
The mice were randomly placed in one of six groups (n=5):
Group (1), mice without treatment (Healthy).
Group (2), healthy mice that received B. fagaroides i.p.
(Healthy Bf ip).
Group (3), healthy mice that received B. fagaroides orally
(Healthy Bf oral).
Group (4), mice inoculated with L5178Y cells (L5178Y).
Group (5), mice inoculated with L5178Y cells and treated with
B. fagaroides i.p. (L5178Y Bf ip).
Group (6), mice inoculated with L5178Y cells and treated with
B. fagaroides orally (L5178Y Bf oral).
On the 17th day, mice were weighed, anesthetized in an ether
chamber, and then sacrificed.
L5178Y lymphoma and body weight gain
The L5178Y murine lymphoma is a thymic tumor conserved
by weekly i.p. transplants in BALB/c mice. It is an excellent
experimental model of lymphoma for its rapid growth inside the
peritoneal cavity, which provokes weight gain, and the absence of
metastatic potential. Therefore, average body weight gain at the
end of the experimental period has proven adequate to follow
lymphoma growth. L5178Y is very aggressive, and BALB/c mice
inoculated i.p. with 20 x 106 L5178Y cells die within 15 ± 2 days
. In the present work, the tumor was induced on the first
day of the experiment by a single peritoneal injection of 2 x 104
ODC radiometric assay
ODC is expressed in small amounts in almost all cells, but its
expression has been reported higher in the kidney [24,25]. In a
recent report, we confirmed higher levels of PA in the kidney than
in the intestine of L5178Y mice . Additionally, in a preliminary
evaluation, the ODC activity showed to be 1000 times higher in
kidney than in the liver, mesothelium, and small intestine (data
not shown); thus, based on these preliminary data kidneys were
used here to evaluate ODC activity. At the moment of dissection,
a fragment of the right kidney was obtained and immediately
processed for ODC enzyme activity assay. The rest of the kidney
was stored at -80 °C for later PA extraction and HPLC analysis.
For the determination of the ODC activity, a crude extract was
prepared from homogenized kidney, as described by Arteaga-
Nieto et al. , except that the buffer was 100 mM Tris-HCl,
pH 7.5. The specific enzymatic activity was assayed using
labeled ornithine (L-[1-14Carboxyl]-ornithine from Amersham
Biosciences, Little Chalfont, Buckinghamshire, England, specific
activity: 1.92 GBq/nmol, concentration 0.56 mM), and measuring
the radioactive 14CO2 generated by ODC. Radioactivity was
measured in a Beckman beta counter and results were expressed
as 14CO2 released in pmols / mg total protein / 90 min. Total
protein concentration was determined according to Bradford
. ODC activity was expressed as pmols 14CO2 released / mg
total protein / 90 min.
Polyamines extraction from kidney
The frozen kidney sample was thawed on ice and homogenized
mechanically in a solution containing 5% trichloroacetic acid
solution, and 0.05N hydrochloric acid (TCA-HCl; 2 μL per mg
of tissue). After centrifugation (10,000 rpm, 20 min, 4°C), the
supernatant was transferred to a fresh tube and stored at -80
°C until analysis. For the chromatographic analysis, 20 μL of the
supernatant, 60 μL of TCA-HCl, and 20 μL of diamine-heptane (0.1
nmol/μL; control peak) were mixed, vortexed, pulse-centrifuged
and injected onto the chromatograph.
Ion exchange Chromatography
The separation of the three main biogenic polyamines:
putrescine (Pu, diamine), spermidine (Spd, triamine), and
spermine (Spm, tetraamine) was based on ion exchange using
a BCX12, 7-10 μm cationic exchange resin (Benson CO, Reno,
Nevada, USA). This method was carried out by HPLC according
to Villanueva and Adlakha , using a Shimadzu fluorometer
(excitation 345 nm, emission 456 nm) and a 100 μL sample
loop automatic injector AS3000 (Spectra-Physics) with
o-phthalaldehyde (OPA, ICN Biomedical, Eschwege, Germany)
post-column detection. All analyses were duplicates. Raw data
visualization, treatment, and analysis were made possible thanks
to the creation of a special extensive object-oriented instrument
controller based on Linux . Data are expressed as pmols/mg
Results are expressed as the mean ± standard error of the
mean (SEM). Data were analyzed by one-way analysis of variance
(ANOVA), and for the comparison of significance between groups
Tukey post-hoc test was used. The probability level of p˂0.05 was
considered significant. Data analyses were performed using the
GraphPad Prism 5.0 software.
Figure 1 presents kidney ODC activity, expressed as pmols
14CO2 release / mg total protein / 90 min. Kidney ODC activity,
increased in L5178Y mice (134.11 ±31.64) compared to healthy
mice (87.59 ±17.44) even though the difference did not quite
reach significance. Remarkably, kidney ODC activity dropped
87% in L5178Y mice that received B. fagaroides i.p. (17.67±9.3,
L5178Y Bf ip group) compared to untreated L5178Y mice.
However, the oral administration of B. fagaroides to L5178Y
mice (L5178Y Bf oral group) did not significantly decrease ODC
activity (95.10±72.97) compared to the L5178Y animals. The
administration of B. fagaroides to healthy mice, either i.p. (96.29
±24.75, Healthy Bf ip) or orally (49.38±20.95, Healthy Bf oral), did
not significantly affect ODC activity.
Body weight gain
The L5178Y mice (L5178Y group) presented a noticeable
increase in body weight compared to healthy mice (Healthy group).
Remarkably the i.p. administration of B. fagaroides maintained
the weight of L5178Y mice to that of healthy animals. However,
the oral administration of B. fagaroides to L5178Y mice lost that
positive effect, and mice from the L5178Y Bf oral group suffered
an important body weight gain, compared to the healthy group,
and similar to that of the L5178Y group of mice. The chronic (15
days) administration of B. fagaroides, either i.p. or orally, did not
affect the body weight of healthy mice. These results coincide with
previous report in the same model .
Correlation between ODC activity and body weight gain
The dispersion plot presented in figure 2 highlights the
relationship between ODC activity and animals’ weight gain
among the different experimental groups. Figure 2 evidences
two clusters: one formed by the groups of healthy mice, the mice
that received B. fagaroides alone (i.p. or orally), and the L5178Y
mice successfully treated with B. fagaroides i.p. This cluster
presented normal body weight, low/mid-ODC activity at the end
of the experimental period, and a good life expectancy. In contrast,
the other cluster is formed by the L5178Y mice and the L5178Y
mice that received B. fagaroides orally. These groups of mice
presented tumor growth (reflected in important weight gain: +
5.2 g and + 4.83 g, respectively), increased ODC activity, and low
Polyamines determination in kidney
As shown in Figure 3, the oral administration of B. fagaroides
(Healthy Bf oral and L5178Y Bf oral groups), significantly increased
putrescine levels in the Healthy (p<0.001) as well as L5178Y
(p<0.01) groups of mice. Meanwhile the i.p. administration of
the plant extract did not present this effect, neither in Healthy
nor in L5178Y mice. The levels of putrescine were similar in
the four groups of mice that did not receive B. fagaroides orally.
No statistically significant variation in kidney spermidine and
spermine content was observed between study groups, except
between the groups of healthy mice that received B. fagaroides
Total protein concentration in kidney
Figure 4 shows that the presence of L5178Y tumor cells
significantly decreased the concentration of total kidney proteins
in the three groups of mice injected L5178Y cells, compared to
animals that did not receive the tumor cells. The administration
of B. fagaroides either i.p. or orally to healthy mice did not affect
ODC activity is a biomarker of L5178Y lymphoma growth.
ODC enzyme is the first rate-limiting enzyme of PA biosynthesis;
it is responsible for the synthesis of the diamine putrescine, the
precursor of the biologically active spermidine and spermine.
ODC activity is essential for normal cell growth; nevertheless, an
increase of its activity beyond a certain threshold exacerbates cell
Our experimental data revealed an elevated kidney ODC
activity in L5178Y mice, which matches with the increase in body
weight due to noticeable peritoneum growth in this group of mice.
However, the levels of spermidine and spermine in the kidneys
did not reflect the increase of ODC activity in this organ. Taking
into account that biosynthesis, transport regulation (uptake and
outtake), and catabolism are important for the maintenance
of PA homeostasis , we hypothesize that the podocytes used
compensatory mechanisms to keep adequate PA levels within
the kidney. These mechanisms probably include the conversion
of excesses of spermidine and spermine back to putrescine (the
precursor of spermidine and spermine), through the activation
of polyamine oxidases, spermine oxidases and acetylpolyamine
oxidases [11,14,20], followed by an enhanced excretion of
putrescine to body fluids, as shown in a previous report in which
we observed high levels of putrescine in the urine of L5178Y mice
. Other mechanisms apart from stimulation of PA conversion
and outtake, may involve a decreased in PA uptake [5,14], or
activation of ODC antizymes to down-regulate ODC expression [2-
In the present work, the data from the Bradford assay showed
that mice injected with L5178Y tumor cells contained lower levels
of total protein in the kidney than the healthy BALB/c mice. This
finding may indicate that the lymphoma growth and the high
ODC activity, affected the kidney’s physiology. As mentioned
above, this tissue presented normal levels of spermidine and
spermine, probably thanks to the activation of oxidases involved
in PA catabolism; these enzymes are a source of reactive oxygen
species (ROS, mainly in the form of H2O2) which increase oxidative
stress and contribute to cellular damage  which may have
induced renal failure . Thus, our findings suggest that in their
intent to keep PA homeostasis through the activation of catabolic
enzymes, the podocytes also contribute to the production of
ROS which in turn provokes renal impairment and proteinuria.
This observation also coincides with an earlier report from de
Blaauw et al. who reported that proteins are chronically wasted
during cancer process in a tumor-bearing rat model induced with
ODC inhibition is a possible mechanism of the
antitumoral effect of B. fagaroides
The main contribution of the present work is to put forward
that the antitumoral effect of B. fagaroides given i.p. involves the
inhibition of kidney ODC activity. We showed that L5178Y mice
treated i.p. with the plant extract presented an 87 % decrease in
ODC activity compared to untreated L5178Y mice. This decrease
coincides with lower levels of urinary putrescine and no weight
gain compared to healthy mice [21,22]. Thus, ODC inhibition by
B. fagaroides prevented the accumulation of PA and therefore
tumor growth. Two mechanisms of actions can be debated. On
one hand, the extract may contain secondary metabolites that
directly inhibit ODC activity as a way to control cell proliferation.
To further prove this point, studies are in progress and the extract
is under chemical characterization in order to isolate and identify
fractions or molecules, in particular triterpenoids and lignins
(podophyllotoxin, pers. com. de JIDS) that have been linked to
reducing the risk of cancer and other major chronic diseases, per
se or in synergistic with other phytonutrients or drugs . On
the other hand, based on the observation of scar-like structure
within the peritoneum as part of the wound healing process
after mechanical action of the needle during the administration
of the extract, we hypothesize that the extract may have favored
the development of fibrosis within the peritoneal cavity and the
formation of a physical barrier that isolated the L5178Y cells
inside the peritoneum (abdominal cavity) and inhibited their
proliferation due to the lack of nutrients.
The administration of B. fagaroides either i.p. or orally to
healthy mice (control mice) for 15 consecutive days did not
significantly affect body weight, ODC activity, spermidine,
spermine or total protein levels, indicating that the plant extract
did not present secondary effects on the global metabolism of
BALB/c mice, especially in kidney.
The evaluation of body weight gain and ODC activity
highlighted the loss of the antitumoral activity when the extract
was given orally. The oral ingestion of the extract and its travel
through the gastrointestinal tract, which is an aggressive
environment, probably affected its biological activity by changing
We also observed that the oral (but not i.p.) administration of
the plant extract significantly increased the levels of putrescine
in kidney of healthy and L5178Y mice. More investigation is now
needed to investigate the relationship between the potential
anti-inflammatory effect of putrescine  and the known
anti-inflammatory effect of the oral administration of Bursera
Kidney ODC activity (this work), and urinary putrescine 
are useful biomarkers of L5178Y lymphoma growth in BALB/c
mice. This is an important point for future projects that foresee
the search for novel cancer therapies that target PA metabolism.
Focusing then our attention on B. fagaroides, we showed that the
antitumoral effect of the crude extract from this native plant of
Mexico, involves, at least in part, the inhibition of ODC activity
in kidney and the control of PA homeostasis. Further work is
now needed to point out the particular groups of metabolites
responsible for this effect.
This study was supported by fundings from the P3E
Program of the Universidad de Guadalajara and the Programa de
Fortalecimiento de la Calidad Educativa - Secretaria de Educación
Pública (PFCE-SEP), Mexico. Our special thanks to PhDs Victor
Raul Villanueva† who inspired this work, and to Pablo Arteaga-
Nieto and Carlos Calvo-Mendez, from the University of Guanajuato,
Mexico, for their support with the ODC protocol.
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